All animals exist in intimate associations with communities of microorganisms, which play important roles in animal development and fitness. The most numerically abundant microbial communities associated with vertebrate animals are the assemblages of microbes in the gut, or the gut microbiota. The mechanisms by which animal-associated microbial communities assemble and persist inside the host are poorly understood. Fundamental unanswered questions about the ecology of animal-associated microbial communities include the extent to which these communities are assembled by host selection or by stochastic processes. If host selection is important, what mechanisms by which this selection occurs? Do selective forces change as a function of host developmental stage? Do the host selective pressures that influence community assembly also contribute to community robustness? An understanding of the assembly principles of these communities is essential for beginning to design therapeutic strategies for humans to safely and effectively promote beneficial microbial communities and prevent or correct pathogenic ones. We propose to apply ecological theory and modeling to understand the assembly and persistence of the gut microbiota in the model vertebrate zebrafish. We have established methods to rear zebrafish under sterile conditions (germ-free) and associate them with defined microbial communities. The zebrafish offers many advantages for these studies. Their fecundity and rapid development allows us to rear thousands of germ-free individuals at a time and design experiments with a high degree of replication. The genetic tractability of zebrafish and their associated bacteria, permit experimental manipulation of host-microbiota associations from both sides of the interaction. The transparency of developing zebrafish allows us to monitor bacterial colonization of the gut in live animals and study spatial and temporal patterns of gut microbiota assembly. An overarching goal of this proposal is to advance the germ-free zebrafish model, through the improvement and standardization of germ-free nutrition and husbandry, and the development of defined zebrafish-associated microbial communities for reductionist analyses, such that this model can become widely used by microbial ecologists and developmental geneticists to study the dynamics of animal-associated microbial communities. In Aim 1, we propose to use ecological theory to calculate the relative importance of host selection and stochastic processes in the assembly of the zebrafish gut microbiota. We will ask whether host selection varies as a function of host developmental stage and microbial taxonomy. In Aim 2, we will determine the relative importance of diet and the immune system in host selection of the zebrafish microbiota. To address the role of diet, we will investigate gut microbiota assembly in fish reared on different diets. To address the role of the immune system, we will analyze gut microbiota assembly in zebrafish mutants with defect innate or adaptive immune systems, and in hosts vaccinated against members of their gut microbiota. In Aim 3 we will investigate the mechanisms that underlie host selection. We will use defined microbial communities to determine the size of the initial colonizing population. We will use in vivo imaging to examine the spatial and temporal patterns of gut colonization. Finally we will determine how diet and immune system contribute to community robustness by monitoring invasion of established microbiota as a function of these host factors. Collectively these studies will provide a deeper mechanistic understanding of how host- associated microbial communities are established and sustained. Such knowledge is essential for the rational design of prebiotics, probiotics, and antibiotics to treat human pathologies associated with imbalances of the microbiota such as inflammatory bowel diseases and diabetes.